Solutions of hemoglobin, derived from human and animal sources, have been proposed as red cell substitutes. While this concept was advanced many years ago, successful application has been hindered by the apparent toxicity of soluble hemoglobin. A meeting of experts, organized by our group and held in March of this year, concluded that hemoglobin is likely to be a reactive species and cause activation of triggered cell and enzyme systems. The platelet was chosen as a test system, with extent and rate of aggregation the end points. Assay of multiple samples with appropriate controls was made possible by use of our multiwell plate system. We tested unmodified stroma-free human hemoglobin, alpha chainlinked human hemoglobin and purified hemoglobin. We found that stroma-free hemoglobin activated platelets and enhanced activation by other stimuli. These effects were not inhibited by anti-oxidants. Platelets were affected to a much lesser extent by the more purified hemoglobins. This result is contrary to a published report that claims not activity at all when platelets were stimulated with purified or unpurified hemoglobin. We believe the discrepancy is the result of technical platelet assay problems in the work leading to the published report. Our conclusions are that the striking reactivity of the stroma free hemoglobin was due to contaminants. We believe that the platelet test system is a valid one and will continue to examine reactions with purified hemoglobins.